anoxic rat heart

ELSEVIER

Neuroscience Letters 220 (1996) 1-4

HEURDSCIENC[ I[T[[IS

Release of the excitotoxic amino acids, glutamate and aspartate, from the isolated ischemic/anoxic rat heart D. Song a, M.H. O'Regan b, J.W. Phillis a'* aDepartment of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA bBiomedical Sciences, School of Dentistry, University of Detroit Mercy, 2985 E. Jefferson, Detroit, All 48207, USA Received 23 September 1996; revised version received 24 October 1996; accepted 25 October 1996

Abstract An isolated rat Langendorff heart preparation has been developed as a model in which to study the release of glutamate, aspartate and other amino acids during i,;chemia, anoxia and hypoglycemia. 15 rain periods of ischemia resulted in large increases in perfusate levels of glutamate, aspartate, glycine, phosphoethanolamine, sefine, alanine, taurine and glutamine. Amino acid levels returned towards preischemic levels in subsequent perfusate collections. Anoxia (15 min duration) increased perfusate levels of most of the measured amino acids, with glutamate and aspartate being particularly affected. In contrast to ischemia, glutamate and aspartate levels declined slowly following reoxygenation. Hypoglycemia (15 min) resulted in small but significantly elevated levels of glutamate and glycine in heart perfusates. As the effects of ischemia or anoxia on glutamate and aspartate release from the heart appear to be comparable to those observed in the brain, it is proposed that the heart preparation may be a suitable model in which to study the ischemia-evoked release of these amino acids in the absence of complications arising from their depolarizing and excitotoxic actions on central neurons. Copyright © 1996 Elsevier Science lreland Ltd.

Keywords: Ischemia; Anoxia; Hypoglycemia; Heart; Glutamate; Aspartate; Excitotoxicity

Cerebral extracellular levels of the excitatory neurotransmitter amino acids, glutamate and aspartate, increase rapidly during ischemic episodes [3,20], contributing to neuronal depolarization, the elevation of intracellular calcium levels and ultimately to neuronal death [2,5]. There is, however, considerable controversy regarding the mechanisms responsible for this increase in the extracellular levels of these excitotoxic amino acids. Depolarization induced, calcium-dependent, exocytotic release of vesicular glutamate and aspartate undoubtedly contributes to the initial efflux, but, being an ATP-dependent process, is unlikely to induce the progressive, continuous, increases in extraceUular glutamate observed during ischemia [16]. Other studies have indicated that a calcium-independent release of glutamate occurs during anoxia or ischemia [13,17]. This type of release may be, in part, a consequence of glutamate release from cytoplasmic stores as a result of a depolarization-induced reversal of the sodiumdependent, high affinity, acidic amino acid plasma mem* Corresponding author. Tel.: +1 313 5776745; fax: +1 313 5775494.

brane transporter [1,17,21]. Yet another suggestion has been that endogenous amino acids are released, in amounts proportional to their concentration gradients, across the cell membrane, as a result of phospholipase-induced plasma-membrane leaks [14]. Definitive studies on central nervous system tissues are, however, complicated by the difficulties of identifying release from neuronal and glial cells, or indeed from the soma, dendrites or synaptic terminals of neurons. Further, as excitatory neurotransmitters, glutamate and aspartate function in a positive feedback link, in which an initial release, by inducing depolarization and increases in intracellular calcium levels, will contribute to a secondary release together with enhanced cell injury. The isolated perfused rat heart was selected as a model in which to study the release of glutamate and aspartate in the absence of appreciable neuronal and glial cell populations. Further, glutamate and aspartate are not cardiotoxic, as is indicated by the beneficial effects on metabolic and functional recoveries of their inclusion (at mM concentrations) in cardioplegic solutions [4,6,18]. The results

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D. Song et al. / Neuroscience Letters 220 (1996) 1-4

reported in this paper describe, apparently for the first time, the release of glutamate, aspartate, glycine, taurine and other amino acids from normoxic, ischemic, anoxic and hypoglycemic rat hearts. Male Sprague-Dawley rats (250-300 g) were anesthetized with intraperitoneal injections of pentobarbital sodium (50 mg/kg). Perfusion of the isolated rat heart with Krebs-Henseleit bicarbonate buffer (KHB) was carried out essentially as described by de Leiris et al. [7] using a constant pressure perfusion system. In brief, hearts were perfused for 15 min for stabilization at a flow rate of circa 5 ml/g per min. In each of the three series of experiments, three basal cardiac perfusate samples were collected at 5 min intervals. Flow to the hearts in the ischemic group was then interrupted for 15 min, after which further samples were collected immediately and at 2, 5, 10, 20 and 30 min. In the anoxia experiments, hearts were perfused with KHB which had been gassed with 95% nitrogen 5% CO2 for several hours (PO2 18-20 mmHg). Perfusate samples were collected at 5, 10 and 15 min after which normal buffer flow resumed. Further samples were collected at 2, 5, 10, 20, 30 and 40 min. For the hypoglycemia experiments, after three control perfusate samples had been collected, the hearts were perfused with glucose-free KHB for 15 min. Samples were collected at 5, 10 and 15 min and then at 2, 5, 10, 20, 30 and 40 min after reperfusion with normal glucose-containing KHB. Collected cardiac perfusate samples were centrifuged at 1200 × g and stored at -20°C. High pressure liquid chromatography (HPLC) assays for perfusate amino acid contents were conducted within a few hours. Statistical differences in amino acid releases within each treatment group were analyzed by ANOVA with contrasts between basal release and the release during subsequent collection periods. A P < 0.05 was accepted as denoting a significant increase over basal levels. Basal heart rates for the Langendorff heart preparations were 221 _+ 11 bpm. Hearts stopped beating at 5.5 + 0.5 min after the onset of ischemia. Exposure to anoxic conditions was associated with a marked reduction in the fre-

quency and strength of contractions. No significant changes in heart rate were observed in the hypoglycemic group. Reperfusion and reoxygenation were accompanied by a recovery of contractile activity, albeit not as strongly as before, in the ischemia and anoxia groups of hearts. All hearts in the ischemia and anoxia groups subsequently developed ventricular fibrillation. No fibrillation was observed in the hypoglycemic group. Basal levels of amino acids in cardiac perfusates are presented in Table 1. The levels of aspartate (270 + 45 nM) and glutamate (361 + 58 nM) were the lowest, with glycine, alanine, taurine and glutamine being present at much higher concentrations. The effects of exposure to ischemia, anoxia and hypoglycemic conditions on all of the measured amino acids, presented as percentage increases above control levels, are also shown in Table 1. For this purpose the initial collection following ischemia has been used for comparisons with the final anoxic and hypoglycemic collections. A comparison of the percent increases in release of all of the amino acids reveals that glutamate and aspartate were the most affected during both anoxia and hypoglycemia. Phosphoethanolamine and glutamate showed the largest percent increases in the initial post-ischemic collections. The effects of the three interventions on aspartate and glutamate levels in the perfusates at individual collection time points are shown in Fig. 1. Both ischemia and anoxia caused large increases in the release of these amino acids, whereas the increases during hypoglycemia were less dramatic. Perhaps the most striking difference between the release patterns was the sustained elevation of glutamate and aspartate levels during the reperfusion period following anoxia in comparison with that following ischemia or hypoglycemia. Studies on the release of glutamate and aspartate in the brain have been complicated by the difficulty of discriminating between metabolic and neurotransmitter (vesicular) pools of these amino acids. Indeed it has been estimated that the larger proportion of extracellular glutamate in the brain (>60%) has a non-transmitter origin [9]. For the

Table 1 Basal and maximal evoked releases of amino acids from the heart Basal (nM/l)

Aspartate Glutamate Glycine PE Serine Alanine Taurine Glutamine

270 361 1912 623 821 5859 2676 11719

+ + + + + + + +

45 58 305 70 62 659 556 1019

% of Basal Ischemia

Anoxia

872 2110 1203 3690 1275 1063 770 804

1497 2392 206 473 217 260 613 146

+ 280* + 631"* _+ 238*** + 1589" + 251"** + 282** + 170"* + 306*

+ + + + + + + +

Hypoglycemia 233*** 662** 59 132" 58 72* 165" 31

273 332 222 230 154 196 128 176

+ 102 + 65** _+ 38* + 58* + 28 + 57 + 19 + 37

Amino acid levels in cardiac perfusates measured just prior to reoxygenation or normoglycemia of the anoxic and hypoglycemic hearts or in the initial perfusate collected from ischemic hearts were compared to those in perfusates collected before exposure to anoxia, hypoglycemia or ischemia. *P < 0.05; **P < 0.01; ***P < 0.001; compared to basal.

D. Song et al. / Neuroscience Letters 220 (1996) 1 - 4

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Fig. 1. Releases of glutamate and aspartate from an isolated perfused rat heart. Line plots show the time course of changes in perfusate amino acids before, during and after 15 min periods (VI) of ischemia (©), anoxia (11) and hypoglycemia(A). Data are presented as means + SEM. Statistically significant increases in perfusate amino acids above basal levels were determinedby ANOVA. *P < 0.05, **P < 0.01, ***P < 0.1301. present investigation, we selected a tissue, the heart, in which aspartate and glutamate are not known to be either excitotoxic or to have a neurotransmitter role, to study ischemia/anoxia-evoked amino acid release. Rather, aspartate and glutamate supplementation improves both the metabolic and functional recovery of the ischemic myocardium [4,6,18]. As in the brain, glutamate is present in rat cardiac myocytes at a higher concentration (2.89 mM) than the other amino acids measured in this study [8]. All of the measured amino acids were present in heart perfusates, indicating that they are released continuously. Under basal conditions, glycine, alanine, taurine and glutamine were present at particularly high levels in the perfusates. Immediately following a 15 min period of ischemia, there were significantly elevated levels of all the amino acids in cardiac perfusates. Two minutes later, the levels of glutamate and aspartate were no longer significantly higher than those of the pre-ischemic controls, although releases of all the other amino acids were still significantly elevated. Anoxia resulted in significant increases in perfusate levels of aspartate, glutamate, phosphoethanolamine, alanine and taurine. In contrast to the other interventions, the levels of glutamate and aspartate declined slowly following reoxygenation (Fig. 1). Hypoglycemia was associated with significant increases in the levels of glutamate, glycine and phosphoethanolamine in cardiac perfusates.

A possible explanation for the slow decline in perfusate glutamate and aspartate levels following anoxia is that there was still sufficient oxygen in the KHB to allow free radical formation in the cardiac myocytes, whereas during ischemia there was a complete cessation of oxygen delivery. Glutamate and aspartate release from the rat cerebral cortex was found to be significantly reduced in a model of complete ischemia versus incomplete ischemia [15]. The present findings with the heart are, therefore, consistent with those studies showing that complete ischemia is less injurious to the brain than is an incomplete ischemia [12,15,19]. These results demonstrate that ischemic, anoxic and hypoglycemic conditions of 15 min duration elicit an increased release of several amino acids, including glutamate, aspartate and glycine which function as neurotransmitters in the central nervous system. In this instance vesicular, synaptic-type release can be excluded, indicating that the amino acids are released from a cytoplasmic pool. It is not clear, at this point, how ischemia, anoxia and hypoglycemia increased amino acid release. Reversal of a high affinity, sodium-dependent, glutamate transporter in depolarized neurons has frequently been proposed as a mechanism for glutamate release from central neurons [1,21], but information concerning the reversibility of amino acid transporters in heart muscle is lacking. Failure of membrane ion pumps, with sodium and chloride accu-

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D. Song et al. / Neuroscience Letters 220 (1996) 1-4

mulation, w o u l d h a v e e v o k e d s w e l l i n g o f the cardiac m y o cytes, w h i c h is k n o w n to result in the release o f glutamate, aspartate, taurine and other a m i n o acids f r o m a variety of cells [10]. E l s e w h e r e [14], we h a v e suggested that g l u t a m a t e and aspartate release f r o m the i s c h e m i c rat cerebral cortex is largely a result o f two m e c h a n i s m s ; reversal o f the glutam a t e transporter and leakage o f a m i n o acids across p l a s m a m e m b r a n e s w h o s e integrity has b e e n c o m p r o m i s e d by the action o f c a l c i u m activated phospholipases. M e m b r a n e disruption, as a result o f p h o s p h o l i p a s e action, c o u l d also account for the early, phase 1, release o f m y o c a r d i a l e n z y m e s o b s e r v e d after 10 m i n o f anoxia from the isolated, perfused rat heart [11]. The isolated, perfused, rat heart, apparently lacking glutamatergic synapses, and on which glutamate has protective, rather than toxic, effects m a y be a suitable preparation in w h i c h to further investigate the m e c h a n i s m s underlying i s c h e m i a - or a n o x i a - e v o k e d a m i n o acid release from excitable tissues. [1] Adam-Vizi, V., External Ca2÷-independent release of neurotransmitters, J. Neurochem., 58 (1992) 395-405. [2] Benveniste, H., The excitotoxin hypothesis in relation to cerebral ischemia, Cerebrovasc. Brain Metab. Rev., 3 (1991) 213-245. [3] Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular ,~oncentrations of glutamate and aspartate in rat hippocampus durin~gtransient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374. [4] Bittl, J. and Shine, K.I., lh'otection of ischemic rabbit myocardium by glutamic acid, Am. J. Physiol., 245 (1983) H406-H412. [5] Choi, D.W., Excitotoxic cell death, J, Neurobiol., 23 (1992) 12611276. [6] Choong, Y.S., Gavin, J.B. and Armiger, L.C., Effects of glutamic acid on cardiac function and energy metabolism of rat heart during ischemia and reperfusion, J. Mol. Cell. Cardiol., 20 (1988) 10431051. [7] de Leiris, J., Harding, D.P. and Pestre, S., The isolated perfused rat heart: a model for studying myocardial hypoxia or ischaemia, Basic Res. Cardiol., 79 (1984) 313-321. [8] Dinkelborg, L.M., Kirme, R.K.H. and Grieshaber, M.K., Transport and metabolism of L-glutamate during oxygenation, anoxia, and

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